这项工作是由美国国立卫生研究院拨款5R01CA100750 - 07 - 04 5R01CA125541，3R01CA125541 - 03S1 - 03 5R01CA129501，3R01CA129501 - 02S1到RS支持

1。大学临床研究协议： <ol><li>两所大学的协议，这一举措的目的。 “第一议定书”允许的肺癌，食管癌，癌瘤，胸腺瘤，间皮瘤患者组织的准采购。该协议还允许从收集生物标志物的研究的患者血液和其他体液。该协议允许研究人员通过抽象的图表，并存储在一个受保护的的数据库中的标本和临床资料等标本的病人源获得的临床资料。 </li><li>第一，除了它使研究人员能够访问以前库存的组织等样品，在病人的癌症诊断和治疗过程中获得的第二个协议是类似。患者同意手术，在美国芝加哥大学，并签署本议定书的资格。 </li></ol> 2。临床数据收集协议： <ol><li>在芝加哥大学医学中心的上述恶性肿瘤的治疗的患者均纳入本议定书。 </li><li>确定了符合条件的患者其治疗肿瘤学家，并在这两个协议的培训的临床团队成员的同意。 </li><li>一旦同意，获得通过图表抽象和一个病人的病历信息输入到数据库中的临床支持团队的成员。 </li></ol> 3。标本采集协议： 组织样本<ol><li>获得通过活检或手术过程中病人的临床护理标准，含有已知或怀疑有恶性肿瘤的组织包括在本议定书。什么是病人的诊断检查所需外，没有额外的组织，获得。 </li><li>样本编制​​的永久部分所需的手术切除后，残留组织被放置在冰上。 </li><li>组织采购技术员运冰病理科残留的样品。 </li><li>残余组织称重，测量，转移到存储，标记，并根据标准作业程序妥为记录。 </li><li>长期存储的标本保存在一个-80℃冷冻病理科。 </li><li>为了获得从已经发生胸部恶性肿瘤手术的患者的组织样本，保持合作的外科医生和放射肿瘤学家的病人名单中引用。在这种方式下，确定了患者的利益。可以检索他们的肿瘤组织样本，从病理学系，如果获得同意。 </li></ol>血液样本<ol start="7"><li>在临床需要抽血，该协议还允许额外的2至6血液5毫升绿盖（肝素）管的管，一个10ML紫色顶管（EDTA的生殖细胞DNA）和一个10毫升红顶管（血清）血。 </li><li>多达6个样品在不同时间点，与血液中的日期绘制注明。 </li><li>在2000年为10分钟的转速离心血液样本。 </li><li>血浆和血清中的组件被分装在1 mL的部分离心管的管。 </li><li>对于收集的白血细胞的接口/红血细胞的一小部分，上部1-2毫升悬浮细胞保存液1-2毫升（MEM EBS的媒体+ 10％的胎儿CAL血清+ 5％DMSO）。 </li><li>所有样品被冻结在异丙基冻结框慢慢在70-80 ° 16-24小时C，然后转移到-70℃至-80 ° C储存盒。 </li><li>所有样品均标有一个条码的唯一标识符和适当的样品采购的形式记录由实验室技术人员。 </li></ol>其他体液： <ol start="14"><li>不供临床使用收集的流体可以根据本协议的收集和储存。为细胞学检查，痰标本收集和发送。痰标本存放在冰在4 ° C，而被转移。 </li><li>痰标本，然后转移至15毫升猎鹰管，并在10分钟1400转离心。 </li><li>上清分装在4毫升6ML cyrovials部分。 Cyrovials被放置在冰箱，干冰，或异丙基冻结框随后冻结到-70℃至-80 ° C。 16-24小时后，样品被转移到-70℃至-80 ° C储存箱。 </li><li>所有样品均标有一个条码的唯一标识符和适当的样品采购的形式记录由实验室技术人员。 </li></ol> 4。建设信息基础设施： <ol><li> Microsoft Access的数据库管理程序进行评估后，被选中作为计划的胸椎到房子临床和实验室数据cology的项目数据库项目根据其可操作性和链接相关的数据集的能力。 </li><li>召开了临床，基础科学的研究人员，生物统计学家，和生物信息学专家小组确定感兴趣的变量捕捉到数据库内。 </li><li>该小组确定有关病人的人口统计，癌症的特性，流行因素和试样注释的基础上胸椎肿瘤研究人员的需求，并指有关的共同数据要素的CDE）由美国国家癌症研究所（设置标准数据元素。 </li><li>该小组开发了一种编码方案，使数据分析的研究目的。在可能的情况下，数据编码使用数字变量，以减少自由文本输入到数据库中的金额。 </li><li>利益的变量分为七个不同的表在Microsoft Access中捕捉到各个方面的相关信息。 </li></ol> 5。设计每个表的内容： <ol><li>七个主要创建表：1）患者的表，2）样本数据表，3）TMA的表，4）DNA样本表，5）胸部肿瘤会议桌，6）细胞株表，和7）C 。 线虫表。 </li><li>患者表被设计成只在数据库中的表房子有关的病人，他们的癌症，其临床过程，及其危险因素，及其结果（图1）的临床相关信息。这个特殊的设计目的是为了限制在数据库中的冗余。 </li><li>他们的病人来源的样本数据表链接的病理标本。每个样品样本病理号，这个数字是关系到病人的医疗记录号码。多个标本可从每一个人，所以，该表还包括标本的日期和标本类型收集的信息：最初的肿瘤，肿瘤复发，或尸检标本。 </li><li>迄今已使用的TMA表捕捉到63个独特的蛋白质的蛋白表达数据。一个TMA的可用于肿瘤和非肿瘤组织中差异表达的蛋白质的分配，使用的抗体，蛋白质（图2）本地化的特点。蛋白表达代​​表的得分为0，1，2，或根据病理学家TMA的染色强度和百分比的印象。 当测量％染色，得分为0表示无染色，1表示低于11％，染色，2表示低于50％，染色，3表示大于50％染色。强度测量时，比分是分配的基础上染色的相对量，也对0到3分。在某些情况下，免疫组化的幻灯片也高分辨率扫描和染色强度自动细胞成像系统（ACIS）在平行病理学家的得分成像软件量化。然而，对于这两种技术，更高的分数表明更大的蛋白表达。 此外，TMA表注释的TMA的打孔位置以供将来参考。此外，该数据库包括源组织（肿瘤，正常，淋巴结，转移组织），样本内的位置（中心，边缘），癌症的组织学，病历号，将此信息链接回患者表。 </li><li> DNA样本表列出了所有的实验室内存储的DNA。 DNA样本，与他们的病人通过病人的病历号来源。有关标本的基本信息被捕获描述范例的来源，包括肿瘤的位置和组织学。本表的目的是描述已内使用聚合酶链反应，DNA测序标准，和突变分析的标本特征的遗传变异。 该表还捕获变量，如氨基酸的改变，核苷酸的变化，纯合子，同义，和在发生突变的基因。已被研究的基因的例子包括Paxillin的，CCBL，表皮生长因子受体，P53，KRAS基因，CMET和EphB4。突变调查还列出。 </li><li>在数据库中的第五个表是胸部肿瘤会议桌。胸部肿瘤会议是一个医疗肿瘤学家，胸外科医生，病理学家，放射科，放射肿瘤学家，胸椎肿瘤临床团队，满足发展协调病人的治疗计划的其他成员的每周例会上。本表的目的是列出那些已经在作为其标准的护理一部分的会议讨论病人。为每一个病人的病理标本的信息也记录在此表。 </li><li>第六表细胞系表。这是一个自由站立在数据库中的表，因为它不与任何其他表。它描述了细胞株已被利用d在实验室用于研究目的。表捕捉氨基酸，核苷酸的变化，纯合子和同义突变，突变细胞系的DNA内的位置。 </li><li>第七表是C。线虫表，它也是一个独立的表。此表列出了同源受体酪氨酸激酶，包括针得分，这是一种与人体蛋白质相似的措施。 </li></ol> 6。建立表之间的关系： <ol><li>每个表都被分配一个主键，作为表内的每个条目的唯一标识符。根据定义，一个唯一标识符值不能重复。例如，在病人表，主键是病历号，因为一个MRN只能意味着一个独特的个体。样本内的数据表，主键是样本病理（SP）的数量。由于TMA和DNA样本表没有唯一标识符，建立一个虚拟的号码作为主键。 TMA和DNA表，再联系到其他表的使用，MRN和SP的号码，分别。这将确保每个TMA的冲床和DNA标本都捐赠病人的临床资料和样本的具体信息链接回。 </li><li>在Microsoft Access中，单个表中的链接通过其主键，这样的逻辑关系建立起来（图3）。这些关系是必要的生成是从多个表中收集数据查询。 </li></ol> 7。查询： <ol><li>在Microsoft Access中执行相关的数据集的查询是相对简单。可以设计一个查询，选择“创建”选项卡下的“查询设计选项。 </li><li>表包括感兴趣的领域的选择和显示。 </li><li>表中的变量选择，如果有必要，可筛选的基础上感兴趣的研究者的标准（图4）。 </li><li>查询，然后可以运行，导致在电子表格的形式列出所需的字段。 </li></ol> 8。导出数据： <ol><li>一旦查询已生成，可以将数据导出。虽然大多数研究人员喜欢的数据在Microsoft Excel电子表格的形式，可将数据导出到其他程序使用“外部数据”选项卡下的出口菜单。可以使用适当的文件扩展名保存数据。 </li><li>当数据用于统计目的的出口，包括在规定的变量，做一个全面的分析，可以使控制协变量的出口。 </li></ol> 9。导入数据： <ol><li>导入数据需要导入的数据格式，并在Access的表格式之间的完美匹配。导入表内利息的变量必须为Access表相同的名称。拼写必须准确，不能有空格的地方空间在Access表中不存在。 </li><li>一旦达到两个表之间的对称，用户有能力使用追加或更新查询到的数据导入到Access。追加查询将允许用户到Access数据库中添加新的数据行。例如，如果没有在数据库中已有的对病人进行，可以使用追加查询添加的病人。但是，如果新的数据是对患者或标本，需要进行修改，更新查询，必须执行这些条目。 </li></ol> 10。更新数据库： <ol><li>数据库项目的成员有规定，以确保该数据库是保持当前的角色。一个全职雇员的工作是负责定期填充和更新已同意在诊所对患者的临床资料数据库。 </li><li>另一个全职雇员，作为数据管理，收费是获得实验室数据，因为它成为可用此信息输入到数据库中更新或追加查询。 </li><li>在协议训练的研究助理，负责执行一个数据库系统的更新，每6个月获得的最新数据。这是特别重要的领域，如重要的地位和最后接触之日起，对数据进行生存分析，因为这些领域有直接影响。 </li></ol> 11。访问数据库： <ol><li>数据库仅是个人谁是HIPAA的要求，并根据IRB协议包括。访问进一步减少在Microsoft Access和有直接责任更新或修改的数据的受过培训的个人。 </li><li>数据库作出了贡献的研究人员可能会要求从数据管理的数据库的信息，但他们本身不允许直接访问数据。 </li><li>数据管理器删除病历号和病人的名字，如变量生成导出查询时，要求研究人员提供的信息去确定。 </li></ol> 12。代表性的成果： 一个研究者可能有兴趣知道的过度表达在非小细胞肺癌的蛋白Paxillin的临床意义。由于这个研究员已产生了很大的Paxillin的数据库中的TMA数据，数据经理批准研究者的请求访问临床与实验室数据信息关联。数据管理器运行一个查询，在那里他结合了患者的表和TMA的表。患者表兴趣的变量患者的出生日期，他们的种族，他们的癌症的组织学，其癌症的阶段，其诊断日期，其重要的地位，其死亡的日期，以及其最后接触日期。使用这些变量，如年龄在诊断和阶段，可占重要混杂因素和控制。从TMA的表，如肿瘤的类型和蛋白表达的重要信息可以被确定。 由于这两个表通过的病历号，病人的肿瘤已Paxillin的表达研究的个人信息联系在一起，是包含在输出。可以进行筛选，以便只与非小细胞肺癌患者显示结果。结果可以进一步完善的基础上，研究者的需求。 这些结果可以由生物统计学家和出口的主要数据进行分析的结果，然后与研究员共享。 项目主页：Access数据库中的模板和标准操作程序是： <a href="http://www.ibridgenetwork.org/uctech/salgia-thoracic-oncology-access-template">http://www.ibridgenetwork.org/uctech/salgia-thoracic-oncology-access-template</a> 许可证：学术和非营利使用免费提供的。 限制使用非学术界：商业用户需要许可证。有关商业用途的问题，请联络（773）702-1692或大学芝加哥的技术和知识产权（UChicagoTech） <a href="www.tech.uchicago.edu">办公室</a> www.tech.uchicago.edu <img src="/files/ftp_upload/2414/2414fig1.jpg" alt="图1" /> 图1。Access数据库截图描绘了病人表的部分。 <img src="/files/ftp_upload/2414/2414fig2.jpg" alt="图2" /> 图2示意图描绘了组织芯片（TMA）2。 <img src="/files/ftp_upload/2414/2414fig3.jpg" alt="图3" /> 图3。截图描绘在Access数据库中的表之间建立了合作关系。表通过主键相联系。 <img src="/files/ftp_upload/2414/2414fig4.jpg" alt="图4" /> 图4。Paxillin的突变，TMA的结果，与临床变量的样本查询。

<ol>
	<li>Adamski, J., Finnegan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Perspectives+on+Microsoft+Office+Access.">New Perspectives on Microsoft Office Access.</a> , (2007).</li><li>Giltnane, J., Rimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+Insight:+Identification+of+biomarkers+with+tissue+microarray+technology.">Technology Insight: Identification of biomarkers with tissue microarray technology.</a> Nat Clin Pract Oncol. 1, 104-111 (2004).</li></ol>

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> </td></tr><tr><td>离心管</td><td>鹘</td><td> 518 - PG </td></tr><tr><td>最低限度基本培养基鹰（MEM） </td><td>西格玛</td><td> M4655 - 500ML </td></tr><tr><td>胎牛血清</td><td> Cellgro胎牛血清您好</td><td> MTT35011CV </td></tr><tr><td>二甲基亚砜（DMSO） </td><td>美国生物分析</td><td> AB03091 </td></tr><tr><td> BD真空采血管血清管</td><td>菲舍尔科学</td><td> 367815 </td></tr></tbody></table>

generation of comprehensive thoracic oncology database - tool for translational research

胸椎肿瘤项目数据库项目创建作为注释癌标本和临床资料内胸部肿瘤研究计划提供给研究人员一个全面，核实，并访问资源库。该数据库还捕获了大量从各种肿瘤组织的研究获得的基因组和蛋白质组数据。召开了临床和基础科学的研究人员，生物统计学家，和生物信息学专家团队来设计数据库。利益的变量作了明确的规定，他们的描述写在一个标准的操作手册，以确保数据注释的一致性。使用准组织银行的协议和其他协议进行回顾性银行，从患者的肿瘤和正常组织样本同意这些协议的收集。抽象，如人口统计，癌症的特性，对这些患者的治疗计划的临床资料，并进入一个Access数据库。蛋白质组学和基因组数据已包含在数据库中，并已与在数据库中所描述的病人的临床资料。从每个表中的数据与使用在Microsoft Access中的“关系”功能，让数据库管理器连接在临床和实验室的信息查询。然后可以查询的数据导出，进行统计分析和假设一代。

Watch this Scientific Journal Video about Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research at JoVE.com

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

胸部肿瘤学数据库的开发作为一个全面的临床和实验室数据转化研究的目的资料库。该数据库将成为平移癌症研究人员在胸部肿瘤研究计划。这个数据库是适应其他癌症模型，以及其他人类疾病。

综合胸部肿瘤生成数据库 - 翻译研究的工具

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

generation-comprehensive-thoracic-oncology-database-tool-for

Research

JoVE Journal

Medicine

15.9K 次观看.  University of Chicago. 胸部肿瘤学数据库的开发作为一个全面的临床和实验室数据转化研究的目的资料库。该数据库将成为平移癌症研究人员在胸部肿瘤研究计划。这个数据库是适应其他癌症模型，以及其他人类疾病。

视频: Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

科研

医学

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

平移血管复合同种异体移植（VCA）研究一种改进的异位猪后肢移植模型

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients&#039; Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

外来体miRNA的分析非小细胞肺癌（NSCLC）患者的血浆通过定量PCR：一种可行的液体活检工具

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

用于疾病建模的人三维肺组织培养 (3D-LTCs) 的生成

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol has been used for the decompression of trapped neurovascular structures in the TOS. Among the other surgical procedures, the advantage of the TFRR is that it has the smallest rate of recurrence and better cosmetic outcomes. The disadvantage of TFRR is that it provides a narrow, and deep working corridor that makes obtaining vascular control challenging.

Here, we present a protocol of the transaxillary resection of the first rib for treatment of thoracic outlet syndrome caused by compression of the brachial plexus, subclavian vein and artery.

经腋窝第一肋骨切除术治疗胸廓出口综合征

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...